EP1358321A1 - Insertionsmutagenesetechnik - Google Patents

Insertionsmutagenesetechnik

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Publication number
EP1358321A1
EP1358321A1 EP02710174A EP02710174A EP1358321A1 EP 1358321 A1 EP1358321 A1 EP 1358321A1 EP 02710174 A EP02710174 A EP 02710174A EP 02710174 A EP02710174 A EP 02710174A EP 1358321 A1 EP1358321 A1 EP 1358321A1
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EP
European Patent Office
Prior art keywords
transposon
cells
transposase
gene
vector
Prior art date
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EP02710174A
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English (en)
French (fr)
Inventor
Charalambos Savakis
Frank Erasmus Universiteit Rotterdam GROSVELD
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Minos Biosystems Ltd
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Minos Biosystems Ltd
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Publication date
Priority claimed from GB0102816A external-priority patent/GB0102816D0/en
Priority claimed from GB0105642A external-priority patent/GB0105642D0/en
Application filed by Minos Biosystems Ltd filed Critical Minos Biosystems Ltd
Publication of EP1358321A1 publication Critical patent/EP1358321A1/de
Withdrawn legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/60—Vectors containing traps for, e.g. exons, promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
    • C12N2830/205—Vector systems having a special element relevant for transcription transcription of more than one cistron bidirectional

Definitions

  • the present invention relates to a method for targeting genes in a cell using a combination of integrating vectors.
  • Such vectors may be viruses and transposons.
  • the method according to the invention comprises the stable provision of a transposase activity, to catalyse transposon mobilisation, in the cell.
  • the techniques described herein are generally useful for genetic research in whole organisms, including animals, for example mammals, including humans, insects, and cells, primary cell cultures and cell lines derived therefrom, and in particular for functional analysis of mammalian genomes.
  • Insertions may have small phenotypic effects, for example resulting from the insertion of a few amino acids into the sequence of a polypeptide or decreased expression of the gene. Alternatively, the effects may be more pronounced, possibly including the complete inactivation of a gene.
  • Insertion events may be detected by screening for the presence of the vector, by probing for the nucleic acid sequence thereof.
  • insertion vectors may be used to upregulate the expression of genes.
  • a vector may be modified to include an enhancer or other transcriptional activation element. Insertion of such a transposon in the vicinity of a gene upregulates expression of the gene or gene locus. This embodiment has particular advantage in the isolation of oncogenes, which may be identified in transformed cells by localisation of the vector.
  • Transposons are genetic elements which are capable of "jumping” or transposing from one position to another within the genome of a species. They are widely distributed amongst animals, including insects. Transposons are active within their host species due to the activity of a transposase protein encoded by the elements themselves. Advances in the understanding of the mechanisms of transposition have resulted in the development of genetic tools based on transposons which can be used for gene transfer.
  • Tc1/mariner family have terminal inverted repeats which end with a highly conserved sequence (CAGTGC). They integrate into the sequence TA and contain a single gene encoding a related polypeptide.
  • CAGTGC highly conserved sequence
  • An alignment of the open reading frames found in the Tc1-like elements has been published by Henikoff (1992) New Biologist 4, 382-388.
  • Other Tc1/mahner elements have been detected by hybridisation, PCR amplification or database searches in different nematode species (Abad et al., (1991) J. Mol. Evol. 33, 251-258; Sedensky et al., (1994) Nucleic Acids Res.
  • Minos is a transposable element of the Tc1 superfamily derived from Drosophila (Franz and Savakis, (1991) NAR 19:6646). It is described in US patent 5,840,865, which is incorporated herein by reference in its entirety. The use of Minos to transform insects is described in the foregoing US patent.
  • Mariner is a transposon originally isolated from Drosophila, but since discovered in several invertebrate and vertebrate species. The use of mariner to transform organisms is described in International patent application WO99/09817.
  • Hermes is derived from the common housefly. Its use in creating transgenic insects is described in US patent 5,614,398, incorporated herein by reference in its entirety.
  • PiggyBac is a transposon derived from the baculovirus host T chplusia ni. Its use for germ-line transformation of Medfly has been described by Handler et al., (1998) PNAS (USA) 95:7520-5.
  • the integration sites are near hypersensitive sites in the host genome (such as DNase 1 hypersensitive sites, and often near transcribed genes) which limits the randomness of insertions.
  • Integration is in principle a recombination process using short homologies between the incoming DNA and the insertion site. Hence there will be a difference between the likelihood of integration at different sites dependent or their accessibility. The latter depends on the state of the chromatin at any site, but also on the type of recombination and the homology in question.
  • Different enzymes are responsible for different integrations: viral integration is controlled by a viral integrase, whilst transposons depend on a cognate transposase.
  • transposon insertion is more random than viral or homologous insertion, it suffers from inefficiencies in transposon mobilisation.
  • transposase in mammalian cells, driven by transposase provided, for example, on DNA vectors.
  • this approach is not demonstrated; in fact, use of DNA vectors to deliver a transposase gene is highly inefficient and transposition cannot reliably be achieved.
  • transposase protein is supplied exogenously to the cell.
  • transposon DNA into cells.
  • methods for introducing plasmid DNA into cultured cells include calcium co- precipitation, lipofection, electroporation and direct injection; results vary considerably with cell line and method.
  • a method for producing a library of genetic mutations in a cell population by insertional mutagenesis wherein a composite vector comprising at least two nucleic acid elements capable of insertion into the cell genome by different mechanisms is used to give rise to two or more mechanistically different insertional events in said cell population.
  • a viral vector comprising a transposable element may be used to effect both viral integration and transposon mobilisation in the cell population, exploiting the ability of the viral and transposon components of the invention to integrate into different parts of the genome with differing frequency.
  • the invention provides a method for producing a library of genetic mutations in a cell population by insertional mutagenesis, wherein a transposon is introduced into the population of cells, which population of cells stably expresses the cognate transposase for said transposon, and the transposon is mobilised to give rise to the genetic mutations.
  • the transposon is preferably delivered using a viral vector.
  • the invention provides a method for producing a library of genetic mutations in a cell population by insertional mutagenesis in which insertion of a vector into a gene leads to gene inactivation, wherein the vector comprises an inducible promoter 5' to the insertion site which drives the expression of an antisense transcript of said gene.
  • both the alleles of the gene may be inactivated; one by insertional deletion, and the second by antisense RNA transcribed from the first allele which contains the inserted vector.
  • the inducible promoter is advantageously a tetracycline promoter (Gossen, M., Freundlich, S., Bender, G., M ⁇ ller, G., Hillen, W. and Bujard, H. (1995) Transcriptional activation by tetracycline in mammalian cells.
  • the vector is a viral vector which encodes one or more transposons.
  • the cognate transposase(s) is or are advantageously stably expressed in the cell population.
  • delivery of the nucleic acids may be accomplished by any available technique, including transformation/transfection, delivery by viral or non-viral vectors and microinjection. Each of these techniques is known in the art. Ribonucleic acids, in particular, may be delivered by microinjection or by viral transduction, particularly by RNA viruses.
  • a method for producing a library of genetic mutations in a cell population by insertional mutagenesis wherein a viral vector comprising a transposon is used to deliver said transposon to said cell population, which cell population stably expresses the cognate transposase for said transposon, and the transposon is mobilised to give rise to the genetic mutations.
  • a viral vector comprising a transposon is used to deliver said transposon to said cell population, which cell population stably expresses the cognate transposase for said transposon, and the transposon is mobilised to give rise to the genetic mutations.
  • viral delivery of a transposon to a cell which stably expresses the cognate transposase gives highly efficient transposon mobilisation.
  • a genetically marked transposon is engineered into the genome of a virus that cannot replicate in the target cells.
  • the virus is packaged, purified, and viral particles are used to infect the target cells. After infection, uncoating and (for RNA viruses) reverse transcription, the transposon DNA is available in the target cell for transposase- mediated transposition into chromosomal sites.
  • the virus may be an integrating or non-integrating virus.
  • the invention moreover provides the advantage of the first aspect thereof, namely, the provision of two mechanistically separate integrating elements in a single composite vector.
  • Figure 1 shows the transposon MiLRgeo, inserted into the first intron of a hypothetical target gene.
  • L and R are the inverted repeats of the Minos transposon.
  • Figure 2 shows generation of a recombinant baculovirus vector carrying a transposon by homologous recombination.
  • Figure 3 shows the BacMiSV40neo transposon virus.
  • pA is the SV40 polyadenylation region and hyd are the Drosophila hydei genomic sequences flanking the original Minos transposable elements.
  • Figure 4 shows the BacCMV/ILMi helper virus.
  • pA is the polyadenylation site of the bovine growth hormone gene
  • Figure 5 shows the pBI-L/ILMi helper plasmid.
  • pA are polyadenylation sites.
  • ILMi is the intronless Minos transposase gene.
  • Figure 6 shows the PBO-MG1 lentiviral vector construct.
  • Figure 7 shows a Southern blot of genomic DNA from clones of MEL cells carrying an integrated copy of the lenti-Minos-GFP virus.
  • the DNA was digested with BspE I and probed with a 3'LTR end fragment probe.
  • Lanes 2 & 4 have DNA from the clones transfected with the plasmid pNT-1 , carrying the CMV driven transposase gene resulting in a transposition that gives a new band that hybridises with end fragment probe.
  • a "cell population” is a population of a suitable cell type in which it is desired to introduce genetic mutations. Suitable cell types are described below.
  • the population is advantageously large in size, and may number anything up to 10 15 or more.
  • it is larger than 100 cells, and preferably larger than 1000 cells, for example 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 cells or more.
  • a cell population may moreover be a non-human animal, preferably a mammal or insect.
  • a “composite vector” is a nucleic acid vector capable of integrating into the genome of a cell which comprises two integrating elements.
  • an “element” is a nucleic acid sequence which is capable of integrating into the genome of a cell.
  • Mechanismically different insertions are insertions which take place by different mechanisms - for example viral integration, transposon integration, homologous recombination and the like.
  • the integration events are catalysed by enzymes.
  • mechanistically different insertional events are catalysed by different enzymes.
  • a nucleic acid may be any nucleic acid, including DNA and RNA, as well as synthetic nucleic acid homologues such as backbone-modified nucleic acids including methylphosphonates, phosphorothioates and phosphorodithioates, where both of the non-bridging oxygens are substituted with sulphur; phosphoroamidites; alkyl phosphotriesters and boranophosphates.
  • Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate.
  • Peptide nucleic acids replace the entire phosphodiester backbone with a peptide linkage.
  • Sugar modifications are also used to enhance stability and affinity.
  • the ⁇ -anomer of deoxyribose may be used, where the base is inverted with respect to the natural ⁇ -anomer.
  • the 2'-OH of the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity.
  • Modification of the heterocyclic bases must maintain proper base pairing.
  • Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine.
  • 5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.
  • a ribonucleic acid may be natural or modified RNA.
  • the RNA may comprise one or more of the modifications identified above.
  • the cell population may be any suitable cell type, including plant, insect and mammalian cells.
  • the cells may be part of an organism, in primary culture, or established cell lines. Mammalian cells including (embryonic) stem cells are preferred.
  • the method of the present invention may be used in transgenic organisms, such as transgenic insects, mammals or plants.
  • cells for use in the methods of the invention may be derived from any source, such as prokaryote, yeast, plant and other higher eukaryote cells.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5 ⁇ and HB101 , or Bacilli.
  • Further host cells include eukaryotic microbes such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae.
  • Higher eukaryotic cells include insect and vertebrate cells, particularly mammalian cells, including human cells, or nucleated cells from other multicellular organisms.
  • vertebrate cells in culture tissue culture
  • useful mammalian host cell lines are epithelial or fibroblastic cell lines such as mouse embryonic stem (ES) cells, Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells.
  • Animal cells include cell lines derived from animals of the phyla cnidaria, ctenophora, platyhelminthes, nematoda, annelida, mollusca, chelicerata, uniramia, Crustacea and chordata.
  • Uniramians include the subphylum hexapoda that includes insects such as the winged insects.
  • Chordates includes vertebrate groups such as mammals, birds, reptiles and amphibians. Particular examples of mammals include humans, non-human primates, cats, dogs, ungulates such as cows, goats, pigs, sheep and horses and rodents such as mice, rats, gerbils and hamsters.
  • Plant cells may be derived from plants including the seed-bearing plants angiosperms and conifers.
  • Angiosperms include dicotyledons and monocotyledons.
  • dicotyledonous plants include tobacco, (Nicotiana plumbaginifolia and Nicotiana tabacum), arabidopsis (Arabidopsis thaliana), Brassica napus, Brassica nigra, Datura innoxia, Vicia narbonensis, Vicia faba, pea (Pisum sativum), cauliflower, carnation and lentil (Lens culinaris).
  • Examples of monocotyledonous plants include cereals such as wheat, barley, oats and maize.
  • transposon may be used in the method of the invention.
  • the transposon is selected from the group consisting of Minos, mariner, Hermes and piggyBac.
  • the transposon is Minos.
  • Each transposon is advantageously employed with its natural cognate transposase, although the use of modified and/or improved transposases is envisaged.
  • the transposon preferably comprises a nucleic acid sequence encoding a heterologous polypeptide.
  • This sequence will be integrated, together with the transposon, into the genome of the cell on transposon integration. Moreover, it will be excised, together with the transposon, when the latter excises on remobilisation.
  • the heterologous polypeptide is a selectable marker. This allows cells having integrated transposons to be identified and the site of integration to be accurately mapped. Transformation efficiency, expressed as percentage of individuals giving transformed progeny, is a crucial parameter in designing strategies for transgenesis, especially for species that are difficult to breed.
  • Mobile element mediated transgenesis is usually based on two components; a transposon and the homologous or cognate transposase.
  • transposases of 7c7, Mos1 and Himarl can also catalyse transposition in vitro (Tosi, L. R. and Beverley, S. M. (2000) Nucleic Acids Res, 28, 784-90; Lampe, D. J., Churchill, M. E. and Robertson, H. M. (1996) Embo J, 15, 5470-9; and Franz, G. and Savakis, C. (1991) Nucleic Acids Res, 19, 6646).
  • transpositional activity may not be proportional to the amount of transposase present; high concentrations of transposase may inhibit transposition in vitro (Lampe, D. J., Grant, T. E. and Robertson, H. M. (1998) Genetics, 149, 179-87) and in vivo (Loukeris, T. G., Area, B., Livadaras, I., Dialektaki, G. and Savakis, C. (1995) Proc Natl Acad Sci U S A, 92, 9485-9; our own results).
  • This work shows that the use of in vitro synthesised Minos transposase mRNA can result in high transformation efficiencies in both species that were tested, D. melanogaster, and the medfly C. capitata.
  • a transformation frequency of 3.2% has been accomplished in Drosophila melanogaster by injecting pMiwl , a non-autonomous Minos transposon marked with a wild-type version of the white gene, to pre-blastoderm embryos carrying a chromosomal source of transposase (Loukeris et al., Op. Cit.). Similar transformation frequencies (ca 1-6%) have been reported for Mnos-mediated transformation of Drosophila, using the same transposon combined with a transposase expressing (helper) plasmid (Loukeris et al., Op. Cit.).
  • transposase levels may vary according to the promoter that drives its expression and, in the latter case, the amount of plasmid injected. Gradual improvements of technique have resulted in increased transformation efficiencies.
  • Mnos-mediated transformation efficiency of up to 10% has been achieved in Drosophila (unpublished data), using various transposons and the helper plasmid that was originally used by Loukeris et al. (Loukeris et al., Op. Cit.). Transformation rates of different insect species may vary widely, depending on the species and the transformation system.
  • a transgenic organism for use in the present invention is preferably a multicellular eukaryotic organism, such as an animal, a plant or a fungus.
  • Animals include animals of the phyla cnidaria, ctenophora, platyhelminthes, nematoda, annelida, mollusca, chelicerata, uniramia, Crustacea and chordata.
  • Uniramians include the subphylum hexapoda that includes insects such as the winged insects.
  • Chordates includes vertebrate groups such as mammals, birds, reptiles and amphibians. Particular examples of mammals include non-human primates, cats, dogs, ungulates such as cows, goats, pigs, sheep and horses and rodents such as mice, rats, gerbils and hamsters.
  • Plants include the seed-bearing plants angiosperms and conifers.
  • Angiosperms include dicotyledons and monocotyledons.
  • dicotyledonous plants include tobacco, (Nicotiana plumbaginifolia and Nicotiana tabacum), arabidopsis (Arabidopsis thaliana), Aspergillus niger, Brassica napus, Brassica nigra, Datura innoxia, Vicia narbonensis, Vicia faba, pea (Pisum sativum), cauliflower, carnation and lentil (Lens culinaris).
  • monocotyledonous plants include cereals such as wheat, barley, oats and maize.
  • transgenic animals are well known in the art. A useful general textbook on this subject is Houdebine, Transgenic animals - Generation and Use (Harwood Academic, 1997) - an extensive review of the techniques used to generate transgenic animals from fish to mice and cows. Techniques for producing transgenic plants are also well known in the art. Typically, either whole plants, cells or protoplasts may be transfected with a suitable nucleic acid construct encoding a binding domain or binding partner. There are many methods for introducing transforming DNA constructs into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods include Agrobacterium infection (see, among others, Turpen et al., 1993, J. Virol.
  • Methods, 42: 227-239) or direct delivery of DNA such as, for example, by PEG-mediated or liposome-mediated transformation, by electroporation or by acceleration of DNA coated particles.
  • Acceleration methods are generally preferred and include, for example, microprojectile bombardment.
  • the viral vector may be a retroviral vector, and may be derived from or may be derivable from any suitable retrovirus.
  • retroviruses A large number of different retroviruses have been identified. Examples include: murine leukaemia virus (MLV), human immunodeficiency virus (HIV), simian immunodeficiency virus, human T-cell leukaemia virus (HTLV). equine infectious anaemia virus (EIAV), mouse mammary tumour virus
  • MMTN Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
  • retroviruses may be found in Coffin et al., 1997, "retroviruses", Cold Spring
  • Retroviruses may be broadly divided into two categories: namely, "simple” and “complex”. Retroviruses may even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in Coffin et al., 1997 (ibid). Host range and tissue tropism varies between different retroviruses. In some cases, this specificity may restrict the transduction potential of a recombinant retroviral vector. For this reason, many gene therapy experiments have used MLV.
  • a particular MLV that has an envelope protein called 4070A is known as an amphotropic virus, and this can also infect human cells because its envelope protein "docks" with a phosphate transport protein that is conserved between man and mouse. This transporter is ubiquitous and so these viruses are capable of infecting many cell types.
  • Replication-defective retroviral vectors are typically propagated, for example to prepare suitable titres of the retroviral vector for subsequent transduction, by using a combination of a packaging or helper cell line and the recombinant vector. That is to say, that the three packaging proteins can be provided in trans.
  • a "packaging cell line” contains one or more of the retroviral gag, pol and env genes.
  • the packaging cell line produces the proteins required for packaging retroviral DNA but it cannot bring about encapsidation due to the lack of a psi region.
  • the helper proteins can package a psi-positive recombinant vector to produce the recombinant virus stock. This virus stock can be used to transduce cells to introduce the vector into the genome of the target cells.
  • the lentivirus group can be divided into “primate” and “non-primate” lentiviruses.
  • primate lentiviruses include human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • lentivirus family and other types of retroviruses are that lentiviruses have the capability to infect both dividing and non-dividing cells.
  • retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
  • lentiviral vectors may advantageously be used in the present invention since lentiviruses are capable of infecting a wide range of non-dividing cells, by contrast to certain other retroviruses that require cell division for stable integration.
  • lentiviral vectors are based on HIV, SIV or EIAV.
  • the simplest vectors constructed from HIV-1 have the complete HIV genome except for a deletion of part of the env coding region or replacement of the nef coding region.
  • these vectors express gag/pol and all of the accessory genes hence require only an envelope to produce infectious virus particles.
  • the accessory genes vif, vpr, vpu and nef are non-essential.
  • HIV-based lentiviral vectors One preferred general format for HIV-based lentiviral vectors is, HIV 5'LTR and leader, some gag coding region sequences (to supply packaging functions), a reporter cassette, the rev response element (RRE) and the 3'LTR.
  • gag/pol accessory gene products and envelope functions are supplied either from a single plasmid or from two or more co-transfected plasmids, or by co-infection of vector containing cells with HIV.
  • the adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate.
  • RNA intermediate There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology all of which exhibit comparable genetic organisation.
  • Human adenovirus group C serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.
  • the adenoviruses/adenoviral vectors of the invention may be of human or animal origin.
  • preferred adenoviruses are those classified in group C, in particular the adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad 12). More preferably, it is an Ad2 or Ad5 adenovirus.
  • Ad2 or Ad5 adenovirus canine adenovirus, mouse adenovirus or an avian adenovirus such as CELO virus (Cotton et al., 1993, J Virol 67:3777-3785) may be used.
  • adenoviruses of canine origin and especially the strains of the CAV2 adenoviruses [Manhattan strain or A26/61 (ATCC VR-800) for example].
  • Other adenoviruses of animal origin include those cited in application WO-A-94/26914 incorporated herein by reference.
  • HSV vectors for use in the invention comprising a polynucleotide of the invention may be derived from, for example, HSV1 or HSV2 strains, or derivatives thereof, preferably HSV1.
  • Derivatives include inter-type recombinants containing DNA from HSV1 and HSV2 strains. Derivatives preferably have at least 70% sequence homology to either the HSV1 or HSV2 genomes, more preferably at least 90%, even more preferably 95%.
  • HSV vectors are to be used for gene therapy in humans the polynucleotide should preferably be inserted into an essential gene. This is because if a vector virus encounters a wild-type virus transfer of a heterologous gene to the wild-type virus could occur by recombination. However as long as the polynucleotide is inserted into an essential gene this recombinational transfer would also delete the essential gene in the recipient virus and prevent 'escape' of the heterologous gene into the replication competent wild-type virus population.
  • Attenuated strains may be used to produce the HSV strain of the present invention, here given as examples only, including strains that have mutations in either ICP34.5 or ICP27, for example strain 1716 (MacLean et al., 1991, J Gen Virol 72: 632-639), strains R3616 and R4009 (Chou and Roizman, 1992, PNAS 89: 3266-3270) and R930 (Chou et al., 1994, J. Virol 68: 8304-8311) all of which have mutations in ICP34.5, and d27-1 (Rice and Knipe, 1990, J. Virol 64: 1704-1715) which has a deletion in ICP27.
  • strains deleted for ICP4, ICP0, ICP22, ICP6, ICP47, vhs or gH, with an inactivating mutation in VMW65, or with any combination of the above may also be used to produce HSV strains of the invention.
  • HSV viruses defective in ICP27 are propagated in a cell line expressing ICP27, for example V27 cells (Rice and Knipe, 1990, J. Virol 64: 1704-1715) or 2-2 cells (Smith et al., 1992, Virology 186: 74-86).
  • ICP27-expressing cell lines can be produced by co- transfecting mammalian cells, for example the Vero or BHK cells, with a vector, preferably a plasmid vector, comprising a functional HSV ICP27 gene capable of being expressed in said cells, and a vector, preferably a plasmid vector, encoding a selectable marker, for example neomycin resistance. Clones possessing the selectable marker are then screened further to determine which clones also express functional ICP27, for example on the basis of their ability to support the growth of ICP27- mutant HSV strains, using methods known to those skilled in the art (for example as described in Rice and Knipe, 1990).
  • HSV strains of the invention comprise inactivating modifications in other essential genes, for example ICP4, complementing cell lines will further comprise a functional HSV gene which complements the modified essential gene in the same manner as described for ICP27.
  • HSV genes may be rendered functionally inactive by several techniques well known in the art. For example, they may be rendered functionally inactive by deletions, substitutions or insertions, preferably by deletion. Deletions may remove portions of the genes or the entire gene. Inserted sequences may include the expression cassette described above.
  • HSV genomic DNA is transfected together with a vector, preferably a plasmid vector, comprising the mutated sequence flanked by homologous HSV sequences.
  • the mutated sequence may comprise deletions, insertions or substitutions, all of which may be constructed by routine techniques. Insertions may include selectable marker genes, for example lacZ, for screening recombinant viruses by, for example, ⁇ -galactosidase activity. Mutations may also be made in other HSV genes, for example genes such as ICPO, ICP4, ICP6, ICP22, ICP47, VMW65, gH or vhs.
  • VMW65 the entire gene is not deleted since it encodes an essential structural protein, but a small inactivating insertion is made which abolishes the ability of VMW65 to transcriptionally activate IE genes (Ace et al., 1989, J Virol 63: 2260-2269).
  • Baculovirus vectors may moreover be employed in the invention.
  • the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is a DNA virus which can be replicate only in cells of certain lepidopteran insects and has been used widely for expression of recombinant proteins in insect cells.
  • Baculoviruses such as AcMNPV have been used recently for introducing heterologous DNA with high efficiency in a variety of mammalian cells, such as a hepatoma cell line and primary liver cells, and endothelial cells (Boyce FM, Bucher NL (1996) Baculovirus-mediated gene transfer into mammalian cells.
  • vectors according to the invention may employ conventional ligation techniques. Isolated viral vectors, plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed vectors is performed in a known fashion. Transposon presence and/or mobilisation may be measured in a cell directly, for example, by conventional Southern blotting, dot blotting, PCR or in situ hybridisation, using an appropriately labelled probe which may be based on a sequence present in the transposon. Those skilled in the art will readily envisage how these methods may be modified, if desired. Marker genes
  • Vectors useful in the present invention are advantageously provided with marker genes to facilitate transposon identification and localisation.
  • Preferred marker genes include genes which encode fluorescent polypeptides.
  • green fluorescent proteins (GFPs) of cnidarians, which act as their energy-transfer acceptors in bioluminescence, can be used in the invention.
  • a green fluorescent protein as used herein, is a protein that fluoresces green light
  • a blue fluorescent protein is a protein that fluoresces blue light.
  • GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, from the sea pansy, Renilla reniformis, and from Phialidium gregariu . (Ward et al., 1982, Photochem.
  • a variety of Aequorea-related GFPs having useful excitation and emission spectra have been engineered by modifying the amino acid sequence of a naturally occurring GFP from Aequorea victoria (Prasher et al., 1992, Gene, 111 : 229-233; Heim et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91 : 12501-12504; PCT/US95/14692).
  • a fluorescent protein is an Aequorea-related fluorescent protein if any contiguous sequence of 150 amino acids of the fluorescent protein has at least 85% sequence identity with an amino acid sequence, either contiguous or non-contiguous, from the wild-type Aequorea green fluorescent protein (SwissProt Accession No. P42212). More preferably, a fluorescent protein is an Aequorea-related fluorescent protein if any contiguous sequence of 200 amino acids of the fluorescent protein has at least 95% sequence identity with an amino acid sequence, either contiguous or non-contiguous, from the wild type Aequorea green fluorescent protein of SwissProt Accession No. P42212. Similarly, the fluorescent protein may be related to Renilla or Phialidium wild- type fluorescent proteins using the same standards.
  • Aequorea-related fluorescent proteins include, for example, wild-type (native) Aequorea victoria GFP, whose nucleotide and deduced amino acid sequences are presented in GenBank Accession Nos. L29345, M62654, M62653 and others Aequorea-related engineered versions of Green Fluorescent Protein, of which some are listed above.
  • P4, P4-3, W7 and W2 fluoresce at a distinctly shorter wavelength than wild type.
  • the present invention is particularly useful in enabling the induction of transposition in whole organisms by introducing the transposon into target cells using a viral system followed by induction of transposition using constitutively expressed or inducible transposase systems.
  • Transposase may be expressed in all tissues.
  • transposase may be expressed in a tissue specific manner, by the use of, for example, tissue specific chromatin opening domains. In this way the tissues in which transposition is induced may be controlled.
  • Transposons and sites from which transposons have been excised, may be identified by sequence analysis. For example, Minos typically integrates at a TA base pair, and on excision leaves behind a duplication of the target TA sequence, flanking the four terminal nucleotides of the transposon. The presence of this sequence, or related sequences, may be detected by techniques such as sequencing, PCR and/or hybridisation.
  • Inserted transposons may be identified by similar techniques, for example using PCR primers complementary to the terminal repeat sequences.
  • the invention allows functional mapping of a genome by permitting precise gene modulation and subsequent detection using transposons.
  • the invention in an advantageous embodiment, allows genes to be ablated by transposon insertion and then specifically identified through the transposon "tag" without requiring costly and time-consuming genetic analyses, and frequently without significant amounts of sequencing. It is a particular advantage of the invention that both alleles of a gene may be inactivated; the transposon advantageously contains an inducible promoter (for example, the tet inducible system) 5' to the splice acceptor, which is induced to make an antisense transcript of the gene in question.
  • the antisense RNA inactivate the RNA from the intact allele resulting in a complete or partial knock out of both alleles of the gene.
  • Upregulation of a gene is achieved by introducing a strong transcriptional enhancer 3' to an internal ribosome binding site coupled to the reporter (such as GFP).
  • a strong transcriptional enhancer 3' to an internal ribosome binding site coupled to the reporter (such as GFP).
  • GFP ribosome binding site
  • Different enhancers would be used for different cell types, for example an immunoglobulin enhancer for B cells).
  • the integration of a transposon at the 3' end of a gene would result in a mRNA which also translates the reporter via the internal ribozyme binding site and upregulate the gene through the enhancer (or LCR type sequence).
  • the two methods can also be combined by including the reverse promoter and a splice acceptor 5' to the IRES. Both knockouts and upregulators would be present in the same library.
  • the invention uses transposons to "mark" genes whose expression is modulated by external stimuli.
  • a cell line which has been exposed to transposon mobilisation with a marked transposon is subjected to treatment with an external stimulus, such as a candidate drug or other test agent, and modulation of the expression of the marker observed.
  • Cells in which the marker is over or under- expressed are likely to have the transposon inserted in or near a gene which is upregulated or downregulated in response to the stimulus.
  • the invention may thus be used to provide in vivo enhancer trap and exon trap functions, by inserting transposons which comprise marker genes which are modulated in their expression levels by the proximity with enhancers or exons. Such applications are described in general in EP 0955364 and known in the art.
  • This approach is useful for the study of gene modulation by drugs in drug discovery approaches, toxicology studies and the like. Moreover, it is applicable to study of gene modulation in response to natural stimuli, such as hormones, cytokines and growth factors, and the identification of novel targets for molecular intervention, including targets for disease therapy in humans, plants or animals, development of insecticides, herbicides, antifungal agents and antibacterial agents.
  • natural stimuli such as hormones, cytokines and growth factors
  • Examples 1 and 2 describe the use of baculovirus for high efficiency introduction of transposons into cells expressing transposase using pMiLRgeo and pMiLRneo respectively.
  • Examples 3 and 4 describes the use of retrovirus vectors for introduction of transposons into cells expressing transposase.
  • Example 1 Use of baculovirus for high efficiency introduction of transposons into cells expressing transposase
  • the Autographa californica nuclear polyhedrosis virus (AcNPV) is the most commonly used virus for expression of heterologous proteins. Vectors are available commercially, for example from Clontech, from whom detailed descriptions thereof may be obtained.
  • Minos exon trap vector pMiLRgeo comprises a gene trap construct, consisting of a splice acceptor site followed by an in-frame fusion of the E. coli beta-galactoside gene with a prokaryotic gene conferring resistance to the antibiotic neomycin (a gene trap fusion referred as geo, Scarnes et al., 1995, Proc. Natl Acad. Sci. USA, 92, 6592- 6596).
  • the geo gene does not contain a translational initiation signal, and expression of the beta-gal and neo phenotypes is dependent upon the insertion of the vector in an intron in the correct orientation, so that splicing generates a fusion mRNA that has necessary translational initiation.
  • the exon trap vector, inserted into an intron, is shown schematically in Figure 1.
  • the exon trap can include any of several reporter genes that are available, such as GFP, available from Clontech, beta-galactosidase, beta-lactamase, beta-glucuronidase and luciferase. It may also include selectable genes, such as genes conferring resistance to neomycin, puromycin and hygromycin. Alternatively, it may comprise a fusion between a reporter and a selectable marker, such as geo that is used in this example.
  • the viral genome is a 134 kbp long double-stranded DNA circle, and direct manipulation of it is difficult.
  • Recombinant baculovirus vectors are, therefore, constructed in two steps.
  • the target sequence in this case the entire transposon, in the form of a restriction fragment
  • the gene is the polyhedrin gene of which the coding sequence has been deleted and replaced with a multiple cloning site (MCS).
  • the transfer vector contains an antibiotic resistance gene and an origin of replication, so that it can be propagated in E.coli cells, but not in insect or other eukaryotic cells.
  • the transposon is cloned between the promoter and the polyadenylation signal of the polyhedrin virus.
  • the transfer vector carrying the transposon is co- transfected into appropriate insect cells (e.g. cell line SF9 from the lepidopteran Spodoptera frugiperda) along with a viral vector. Double recombination between the transfer vector and the viral vector results in a recombinant virus containing the transposon (figure 2).
  • the transfer vector contains a complete version of an essential viral gene downstream from the MCS.
  • the viral vector is engineered so that the essential gene is mutated. Double recombination restores function of this gene and provides a strong genetic selection for recombinant virus.
  • transposase is required to mobilise a transposon from its original position on the viral DNA to new chromosomal positions. Continued expression of transposase after integration of the transposon is undesirable because it will lead to re-mobilisation of the transposon.
  • a two-transgene scheme will be employed: Cells are stably transgenic with two constructs: one containing the transposase gene under the control of an activatable promoter and a second containing a stably expressed gene encoding the inducible transcriptional activator of said promoter.
  • a widely used system of this kind in mammalian cells is the tetO promoter-operator, combined with the tetracycline/doxycycline-repressible transcriptional activator tTA, also called Tet-Off gene expression system (Gossen, M. & Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline responsive promoters. Proc. Natl. Acad. Sci. USA 89:5547-5551), or the doxycycline-inducible rtTA transcriptional activator, also called Tet-On system (Gossen, M., Freundlich, S., Bender, G., Muller, G., Hillen, W. & Bujard, H. (1995) Transcriptional activation by tetracycline in mammalian cells. Science 268:1766-1769).
  • Tet-Off gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the culture medium.
  • Tc tetracycline
  • Dox doxycycline
  • Tet-On procedure for establishing cell lines carrying the transcriptional activator gene and the Tet-regulatable gene stably integrated in its chromosomes have been desc ⁇ bed. For example see http://www.clontech.com/techinfo/manuals/PDF/PT3001-l .pdf.
  • the Tet-On system is employed for tetracycline-inducible expression of Minos transposase in a mammalian cell line.
  • a doubly transgenic line is generated by standard illegitimate recombination technology.
  • Two constructs are used: First, a construct containing the rtTA gene under a constitutive promoter expressed in the target cells.
  • An example of such construct is the pTet-On plasmid (Clontech) which contains the gene encoding the rtTA activator under control of the Cytomegalovirus immediate early (CMV) promoter.
  • CMV Cytomegalovirus immediate early
  • the rtTA transcriptional activator encoded by this construct is active only in the presence of Doxycycline.
  • the second construct contains the Minos transposase gene under control of the tetracycline-response element, or TRE.
  • the TRE consists of seven direct repeats of a 42-bp sequence containing the tet operator (tetO), and is located just upstream of the minimal CMV promoter, which lacks the enhancer elements normally associated with the CMV immediate early promoter. Because these enhancer elements are missing, there is no "leaky” expression of transposase from the TRE in the absence of binding by rtTA.
  • An example of such construct is the pTRE2 plasmid (Clontech) in the MCS of which is inserted the gene encoding Minos transposase.
  • rtTA is expressed but does not activate transcription of Minos transposase unless Doxycycline (0.1-1 micrograms/ml) is added in the medium.
  • Doxycycline 0.1-1 micrograms/ml
  • transposon-loaded recombinant baculovirus is used to infect the doubly transgenic cells at high titres, to achieve infection of individual cells by multiple copies of the virus.
  • the virus cannot replicate in mammalian cells, but its DNA moves into the nucleus and has been shown to be accessible to the transcriptional apparatus.
  • cells are exposed to appropriate concentrations of Doxycycline to induce expression of transposase.
  • cells are moved to a medium without doxycycline to arrest transposase expression, and supplemented with appropriate antibiotic (G418 for this example) to select for cells that have the transposon inserted.
  • G418 selection will select only cells that contain an "active" exon trap, i.e. the transposon has inserted into an intron of an expressed gene in such a way that an active neo protein is expressed as result of splicing.
  • Example 2 Use of baculoviruses to introduce transposons into eukaryotic cells.
  • transposable elements as genome-wide insertional mutagenesis agents can be limited by low transfection rates of DNA into cells.
  • One possible way to overcome this obstacle is to use a high-infectivity virus as a vehicle to introduce transposons into cells.
  • a high-infectivity virus as a vehicle to introduce transposons into cells.
  • Recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) was constructed containing a Minos transposon carrying an antibiotic resistance marker gene.
  • Recombinant virus was used to infect a cell line in the presence and absence of transposase and numbers of stably transformed colonies were determined after selection with antibiotic. The presence of transposase resulted in 200-400fold stimulation of stable integration of the transposon. Southern analysis showed that individual colonies carried 1-7 copies of the transposon integrated by transposase- mediated events. In a separate line of experiments, infectivity of baculovirus was tested in a number of mammalian cell lines using a recombinant AcNPV expressing
  • GFP Green Fluorescent Protein
  • transgenic mice and cell lines inducibly expressing transposase are useful for inducing transposition of cognate transposons that are already integrated in chromosomes or that reside on episomal DNA, such as recombinant baculovirus
  • Transgenic mice were generated carrying a construct comprising the Minos transposase under the control of the tet operator (Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551). Expression of transposase can be regulated by doxycycline in double trangenic mice carrying this construct and a gene encoding the tTA or rtTA transcriptional activators (Gossen, M., Freundlich, S.,
  • Double transgenics can be used directly for in vivo transposition experiments, of for generation of primary or immortalized cell lines (Cascio SM. (2001) Artif Organs. 25: 529-538).
  • C. Cell lines inducibly expressing transposase can be generated by stable integration of two constructs: A construct encoding an inducible transcriptional activator (such as the tet activator rtTA) and a construct encoding transposase under the control of the tet operator (tetO).
  • Plasmids containing these constructs were tested for tetracycline-regulatable expression of transposase by co-transfecion into HeLA cells with a plasmid containing a Minos transposon. Treatment with doxycycline increased transposon integration rates 26-fold over non-treated controls.
  • the constructs encoding tet activator and tetO-controlled transposase are used to generate cell lines with stable chromosomal insertions, which can produce transposase inducibly.
  • Recombinant baculovirus and plasmids Recombinant AcNPV baculoviruses were generated using the BacPac Baculovirus Expression System (Clontech) according to manufacturer's instructions (http://www.clontech.com/techinfo/manuals/PDF/PT1260- 1.pdf). Recombinant plasmids were constructed by standard methodologies.
  • BacMiLRneo ( Figure 3) is a recombinant AcNPV virus containing a transposon that consists of the Minos inverted repeats (block arrows) flanking the neo gene under the control of the early SV40 promoter.
  • a Minos transposon containing the SV40neo gene was subcloned as a Kpnl-Sacl fragment from plasmid pMiLRneo (Klinakis, A.G., L. Zagoraiou, D.K. Vassilatis and C. Savakis (2000). EMBO Reports. 1 : 416- 421) into the respective sites of the pBacPAK9cloning vector (Clontech).
  • BacCMV/ILMi ( Figure 4) is a recombinant AcNPV virus containing the processed (intronless) gene encoding Minos transposase under the control of the CMV early promoter.
  • CMV/ILMi was subcloned as a Pvull-Nrul fragment from plasmid pCMV/ILMi (Zagoraiou L, D.
  • pPBI-L/ILMi • pPBI-L/ILMi ( Figure 5) is a helper plasmid based on the pBI-L cloning vector (Clontech). It contains a bidirectional promoter consisting of two copies of a minimal
  • TRE Tetracycline Response Element
  • the TRE consists of 7 direct repeats of a 42 bp sequence containing the tet operator (tetO).
  • Plasmid pPBI-L/ILMi was constructed by cloning the intronless transposase gene from plasmid pHSS6hslLMi20 (Klinakis, A.G., T.G. Loukeris, A. Pavlopoulos and C. Savakis (2000) Insect Mol. Biol. 9:269-275) as a Clal-Sall fragment into the respective sites of pBI-L.
  • HepG2 cells were seeded on six-well plates (750000 cells per well) containing 2 ml of medium (DMEM, Gibco BRL). The medium was replaced one day later and baculovirus was added in a small volume of PBS. In superinfection experiments the second virus was added 4 hours after the first. The virus was removed 15 hours post infection by replacing the medium. The cells were trypsinised 48 hours post infection and 1/2 and1/20 of them were seeded on 60mm plates. Treatment with G418 started 72h post infection. After 25 days, the neo resistant colonies were isolated and propagated in G418 containing medium.
  • medium DMEM, Gibco BRL
  • Transfection of cultured cells with plasmid DNA and selection of stably transfected cells was performed as described previously (Klinakis, A.G., L. Zagoraiou, D.K. Vassilatis and C. Savakis (2000) EMBO Reports. 1: 416-421). Results
  • a recombinant baculovirus that carries a green fluorescent protein cassette (van Loo ND, Fortunati E, Ehlert E, Rabelink M, Grosveld F, Scholte BJ. (2001) J Virol. 75: 961-970) was used to infect several cell lines at a multiplicity of infection (MOI) of 200. Infection efficiencies varied between approximately 20% in the human breast cancer lines MCF7 and T47D, 50% in the human hepatoma HepG2 line and 80% in the rat embryonic fibroblast Ref1 line.
  • MOI multiplicity of infection
  • prtTAM2 (Clontech) containing a rtTA activator expression cassette (the rtTA activator is inactive in the absence of inducible by tetracyclin or doxycyclin)
  • Stably transfected cell lines carrying the tet activator expression cassette from plasmid prtTAM2 and the tetO transposase/luciferace cassette from plasmid pBIL/ILMi is generated by standard procedures.
  • a stable cell line expressing tet inducible transposase is infected with recombinant baculovirus carrying a transposon.
  • Transposase is induced transiently by treatment with doxycyclin and catalyzes transpositions from the episomal viral DNA into chromosomes. Removal of inducer results in removal of transposase and stabilizes insertions of the transposons.
  • Cells carrying stable integrations of the transposon are then selected. Selection is based on selectable or screenable markers carried by the transposon (e.g. antibiotic resistance or expression of autofluorescent proteins).
  • trangenic lines were generated by standard procedures carrying the the tetO transposase/luciferase cassette from plasmid pBIL/ILMi.
  • Double transgenics that inducibly express transposase can be generated by crossing these mice with transgenics carrying a rtTA (or a tTA) expression cassette.
  • Retrovirus vectors for introduction of transposons into cells expressing transposase
  • the transposon is cloned into a retroviral/lentiviral vector by standard recombinant DNA techniques (see e.g. Hoeflich et al 2000, nature 406, p82, where a ⁇ globin cassette is exchanged for a lacZ cassette in an existing retroviral vector).
  • the recombinant transposon/viral vector plasmid DNA is isolated by standard procedures and transfected into the viral packaging cell line as described (Hoeflich et al., 2000). Virions are collected and concentrated as described by Gallardo et al., 1997 (Blood, 90, 952-57).
  • Target cells are infected in the presence of polybrene (8 ⁇ g/ml) as described by Sadelain et al., (PNAS 92, 6728-32, 1995) to establish the starting population of cells containing a transposon insertion after viral integration.
  • the starting population of target cells for infection are either established cell lines, primary cell cultures (from mouse or human or other animals; e.g.. Methods in Enzymology Vol 58, Cell Culture (1979), Academic press Inc. San Diego. Editors W.B. Jakoby and I.H. Pastan, Editors in chief: S.P. Colowick and N.O.) or immortalised cells (e.g. Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larsen L, Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci U S A.
  • Each of these target cells is first transfected with (or infected with a retrovirus containing) a construct containing a Tet inducible (Baron U., Nucl Acid Res., 25, 2723-29, 1997 and references cited therein), or tamoxifen inducible transposase [a modified oestrogen receptor domain (Indra et al., Nucl Acid Res.
  • transposase which retains it in the cytoplasm until tamoxifen is given to the culture
  • a RU418 inducible transposase (operating under the same principle as with the glucocorticoid receptor; see Tsujita et al., J. Neuroscience, 19, 10318-23, 1999).
  • the gene for the transposase in introduced by standard transfection methods or is present in the transgenic animal from which the primary cells are isolated.
  • the transposase gene is introduced into those animals either via microinjection of fertilised eggs by standard procedures (Manipulating the mouse embryo, Hogan et al., Cold Spring Harbor Press, 1994) or introduced into embryonic stem cells via homologous or non homologous recombination.
  • the ES cells are injected into blastocysts to obtain transgenic animals via standard procedures (Manipulating the mouse embryo, Hogan et al., Cold Spring Harbor Press, 1994).
  • the transposon construct is introduced into mice via microinjection of fertilised eggs (Manipulating the mouse embryo, Hogan et al., Cold Spring Harbor Press, 1994) or embryonic stem cells (Manipulating the mouse embryo, Hogan et al., Cold Spring Harbor Press, 1994).
  • the transposon is made to jump in mouse somatic tissues to either isolate somatic cells with different transposon integration or/and germ line tissue (preferably sperm) to establish a population of mice in the next generation that contain the transposon in different positions.
  • transposon via a retroviral infection step (using lentivirus or retrovirus vectors, as above) which establishes a starting population of different germ line insertions. Inducing transposase in the infected germ cells will increase the population of transposons, which is spread by breeding.
  • Transposition is monitored by northern blot analysis, PCR or FISH.
  • Example 4 Use of a Lentivirus vectors for introduction of transposons into cells expressing transposase
  • the construct pBO-MG1 ( Figure 6) is a self-inactivating lentiviral vector plasmid containing Minos transposon sequences flanking the GFP gene driven by the CMV promoter. Plasmid pBO-MG1 was obtained by subcloning the Xho I insert fragment of the plasmid pMiCMVGFP, into the Xho I site of the plasmid pBO2. The parent plasmid pBO2 was derived from the plasmid CS-CG (Myoshi et al. (1998) J. Virol 72, 8150-57).
  • Transposon MiCMVGFP is constructed as follows: The plasmid pMILRTetR (Klinakis et al. (2000) Ins. Mol. Biol. 9, 269-275 (2000b) is cut with BamH I and re-ligated to remove the tetracycline resistance gene between the Mnos ends, resulting in plasmid pMILR ⁇ SamH An /4sp718/Sacl fragment from pMILR ⁇ Sam H1 , containing the Mnos inverted repeats and original flanking sequences from D. hydei, is cloned into plasmid pPolylll-l-lox (created by insertion of the loxP oligo:
  • pMiCMVGFP figure 1
  • the final construct (pMiCMVGFP, figure 1) used for the generation of transgenic mice is created by inserting into the Spe I site of ppolyMILR ⁇ SamHI the 2.2 kb Spel fragment from plasmid pBluescriptGFP, containing a humanised GFP gene (from Clontech plasmid pHGFP-S65T) driven by the CMV promoter and followed by the SV40 intervening sequence and polyadenylation signal.
  • Plasmid pNT-1 A 1 kb Clal Not I fragment containing Mnos transposase cDNA was cloned into Cla / Not of Pev3 (Clare Gooding, Biotechnology Dept, Zeneca, Macclesfield, UK). A 3.8 kb Clal Asp 781 fragment from the resulting plasmid (containing minos transposase cDNA followed by an intron with RNA splicing site and a polyadenylation signal from the human ⁇ globin gene) was subcloned into Cla lAsp718) of the pBluescript SK (Stratagene, La Jolla, Ca, USA) creating the plasmid pBlue/transposase/3 ' ⁇ . Plasmid pNT-1 was derived from the plasmid pBlue/transposase/3' ⁇ by cloning a 580bp blunt ended Sac I- Spe I fragment, spanning the CMV promoter, into the EcoR V site.
  • pMDLg/RRE expresses the HIV-1 gag and pol proteins.
  • pRSV.REV expresses the HIV-1 REV protein.
  • pMD.G expresses the envelope G-glycoprotein of VSV (Vesicular Stomatitis Virus). Details relating to pMDLg/RRE plasmid , the pRSV.REV plasmid and the pMD.G plasmid can be found in Dull, T., Zufferey, R., Kelly, M., Mandel, R.J., Nguyen, M., Trono, D. and Naldini, L. (1998) J. Virol, 72, 8463-8470.
  • All plasmids were prepared by using the Qiagen Endotoxin-Free Maxi- or Giga-prep kit.
  • Human Embryonal Kidney (HEK) cell line 293 T was used for the packaging of lentiviral vectors (Dull et al. 1998).
  • the packaging line 293T was grown to 70% confluence in 10 mis of DMEM-10% FCS in 20 x 10cm dishes.
  • BBS (2x BBS (1 litre ) comprises dissolved NaCI 16.36 g, BES (N,N-bis-(2-Hydroxyethyl)-2- aminoethanesulfonic acid) 10.65 g [from Calbiochem #391334], Na 2 HPO 4 0.21 g in 900 ml H 2 O, titrated to pH6.95 with 1M NaOH and brought to 1 litre with H2O, filter sterilized and stored frozen at -20°C)
  • Virus particles were concentrated by spinning in Beckman SW28 rotor at 19.4K rpm for 2hrs. at room temperature. The pellet was resuspended in 1 ml Hanks Balanced Salt Solution (HBBS) and re-spun in a Beckman SW55 rotor at 21 K for 2 hrs. at room temperature.
  • HBBS Hanks Balanced Salt Solution
  • the viral pellet was suspended in 200 ⁇ l HBBS and vortexed at low speed for 1-2 hrs at room temperature.
  • the suspension was spun in a microfuge and the supernatant stored as 10-50 ⁇ l aliquots at -80°C.
  • Virus was harvested from the 293T producer cell line and used for a PCR assay to determine viral titres.
  • the target cell line (MEL- Murine Erythroleukemia) was seeded at subconfluent concentrations of 1x10 5 cells/ml and allowed to grow O/N at 37°C, 5% CO 2 .
  • the virus was suitably diluted to give an overall MOI of 50 and added to the medium. Incubation with the virus was O/N under the conditions described above.
  • the cells were harvested and cloned by limiting dilutions. Clones positive for the transgene were expanded and single-copy clones transfected with the plasmid p- NT2 harboring the transposase, driven by the CMV promoter, using the transfection reagent "Superfect" (Qiagen # 301305) and incubated O/N under the above conditions. 5) Cells were harvested 48 hrs. following the introduction of the transposase and genomic DNA prepared for analysis by southern blotting.
  • Genomic DNA was digested with Bspe I, which has a single site in the 3' LTR and probed with an end DNA fragment probe.
  • Figure 7 shows a Southern blot of genomic DNA from clones 1 and 2 of MEL cells carrying an integrated copy of the lenti-Minos-GFP virus.
  • the DNA was digested with BspE I and probed with a 3'LTR end fragment probe.
  • Lanes 2 & 4 have DNA from the clones transfected with the plasmid pNT-1 , carrying the CMV driven transposase gene resulting in a transposition that gives a new band that hybridises with end fragment probe.

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